Hyperhomocysteinemia is a modifiable, independent risk factor for cardiovascular disease, cognitive dysfunction, complications of pregnancy, and osteoporosis. Homocysteine-induced endothelial cell dysfunction may link this diverse group of pathologies. Our research has focused on: 1) the vascular biochemistry of homocysteine;2) the mechanism of homocysteine transport in cultured human aortic endothelial cells;and, 3) the molecular targeting of protein cysteine residues by homocysteine. Thiol-disulfide exchange accounts for the forms of homocysteine in circulation. Cysteine transporters mediate homocysteine import in the vascular endothelium. Evidence is provided that links homocysteine targeting to vascular cell dysfunction. The long-term objectives are to understand the mechanism of homocysteine-accelerated atherogenesis and to develop improved interventional strategies that will lower plasma total homocysteine in high-risk subjects and in subjects who are refractory to conventional therapy (e.g., end-stage renal disease). Our central hypotheses are: 1) homocysteine is a mediator of early atherogenesis but becomes a marker in advanced atherosclerosis (we call this the "mediator ?>? marker hypothesis" of hyperhomocysteinemia);2) monocyte chemoattractant protein 1 and other pro-inflammatory cytokines are induced by homocysteine in early atherogenesis by a mechanism involving the activation of NF-kB;3) the fundamental mechanism underlying homocysteine causality is molecular targeting;and, 4) homocysteine-lowering by primary intervention will reduce mortality and morbidity associated with hyperhomocysteinemia. Our specific aims are: 1) to study the mechanism of protein homocysteinylation and functional consequences;2) to study the transport and metabolism of homocysteine and its disulfide congeners in vascular cells;and, 3) to identify intracellular proteins (e.g., metallothionein) targeted by homocysteine and how loss of function of targeted proteins leads to vascular cell dysfunction. The program to fortify the American diet with folic acid, completed in January, 1998, has had a profound effect on reducing the incidence of hyperhomocysteinemia, neural tube defects and, possibly, mortality due to stroke and heart attack. Additional public health benefits are likely by gaining a better understanding of homocysteine transport, metabolism and the adverse effects of homocysteine on our circulatory system.